Emulsion compositions and methods of their use

ABSTRACT

Emulsion compositions are provided herein. Also provided herein are kits containing one or more emulsion compositions or components for making such emulsion compositions. Also provided herein are methods of using such emulsion compositions, such as for amplification of target nucleic acids in emulsion droplets.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/921,453, filed Jul. 6, 2020, which a continuation of U.S.patent application Ser. No. 15/882,098, filed Jan. 29, 2018, now U.S.Pat. No. 11,118,217, which claims benefit of priority to U.S.Provisional Patent Application No. 62/452,330, filed Jan. 30, 2017, eachof which is incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Microfluidic processes often employ the use of an emulsion, whichcontains droplets (herein referred to as “emulsion droplets”) of adispersed liquid phase surrounded by an immiscible continuous liquidphase. Emulsion droplets may be used as reaction vessels for chemical orbiological reactions, as storage vessels, and/or as a method to isolateand compartmentalize molecules, such as chemical or biological elements.A population of emulsion droplets (herein referred to interchangeably asa “population” or “library”) may be composed of droplets of manydifferent sizes (polydispersed population) or droplets of relativelysame size (monodispersed population). With proper chemistry includingpolymers, amphiphilic compounds, surfactants, and the like on thesurface of the emulsion, droplets may be made “stable,” meaning they aresubstantially prevented from mixing and merging when in contact witheach other. This stability allows one to create a population or libraryof droplets composed of different chemical or biological components thatmay be stored in the approximately same volume of space without mixingor contamination between and/or among the components of one droplet andanother. This property of stabilized emulsion droplets is useful formany applications in performing chemical and biological reactions,storage and compartmentalization.

One approach for stabilization of emulsion droplets is to include across-linkable agent (e.g., a cross-linkable protein) in the emulsion.After droplet generation, the cross-linkable agent can be treated toinitiate cross-linking, thereby forming a solid “skin” at the dropletinterface. See, for example, U.S. 2011/0027394 and U.S. 2011/0217712.However, these cross-linkable agents can increase the propensity ofsurface fouling or clogging in microfluidic droplet handling devices,and can inhibit droplet merging and/or injection techniques.

BRIEF SUMMARY OF THE INVENTION

Described herein are stable water-in-oil emulsion droplet compositions,and methods and compositions for forming and using stable emulsiondroplets, including but not limited to microfluidic methods foranalyzing biological analytes. The stable water-in-oil emulsion dropletcompositions typically include an aqueous phase that comprises acyclodextrin and a polymer, wherein at least a portion of thecyclodextrin and polymer in the aqueous phase form a host-guest complex.Without wishing to be bound by theory, it is believed that in someembodiments the stabilization is due to formation by the host-guestcomplexes of a thermally reversible hydrogel. The thermally reversiblehydrogel can be gelate (gel) phase at high temperature and a solution(sol) phase at low temperature. For example, the hydrogel can be gel ata PCR annealing temperature such as 50° C., a PCR extension temperaturesuch as 72° C., a PCR denaturation temperature such as 95° C., or acombination two or more thereof. Additionally or alternatively, thehydrogel can be sol at, e.g., room temperature or below. In someembodiments, at least a portion of the host-guest complexes formed inthe aqueous phase of the water-in-oil form a rotaxane, a pseudorotaxane,a polyrotaxane, or a polypseudorotaxane.

In one aspect, the present invention provides an emulsion comprising awater-in-oil emulsion droplet, the droplet comprising: water; aplurality of cyclodextrin molecules; and a plurality of polymermolecules, wherein individual polymer molecules of the pluralitycomprise a hydrophobic moiety and/or a hydrophilic moiety, wherein theaqueous phase of the droplet comprises a thermally reversible hydrogel,and wherein the droplet is present in a continuous oil phase. In somecases, the thermally reversible hydrogel is hydrogel at a temperatureabove a gel sol transition temperature and liquid at a temperature belowthe gel sol transition temperature. In some embodiments, the thermallyreversible hydrogel is in a gel form at a temperature of at least about60° C. or 70° C. and no more than about 98° C. and a sol form attemperature of less than about 50° C. or 30° C. and greater than about0° C.

In one aspect, the present invention provides an emulsion comprising awater-in-oil emulsion droplet, the droplet comprising: water; aplurality of cyclodextrin molecules; and a plurality of polymermolecules, wherein individual polymer molecules of the pluralitycomprise a hydrophobic moiety and/or a hydrophilic moiety, wherein atleast a portion of the cyclodextrin molecules and the polymer moleculesform a plurality of polypseudorotaxane complexes, and wherein thedroplet is present in a continuous oil phase.

In one aspect, the present invention provides an emulsion comprising awater-in-oil emulsion droplet, the droplet comprising: water; aplurality of cyclodextrin molecules; and a plurality of amphiphilicpolymer molecules, wherein individual amphiphilic polymer molecules ofthe plurality comprise a hydrophobic moiety and a hydrophilic moiety,wherein at least a portion of the cyclodextrin molecules and theamphiphilic polymer molecules form a plurality of polypseudorotaxanecomplexes, and wherein the droplet is present in a continuous oil phase.

In some embodiments, individual cyclodextrin molecules of the pluralityare independently selected from the group consisting of anα-cyclodextrin, and a β-cyclodextrin. In some embodiments, individualcyclodextrin molecules of the plurality are independently selected fromthe group consisting of an α-cyclodextrin, and a γ-cyclodextrin. In someembodiments, individual cyclodextrin molecules of the plurality areindependently selected from the group consisting of a β-cyclodextrin,and a γ-cyclodextrin. In some embodiments, at least a portion ofindividual cyclodextrin molecules of the plurality independentlycomprise a hydrophobic modification.

In some embodiments, individual cyclodextrin molecules of the pluralityare independently a compound of Formula I:

wherein: each R1 is independently selected from the group consisting ofa hydrophobic group, a sulfite, and H, where the hydrophobic group isselected from alkyl, alkenyl, alkynyl, haloalkyl, cycloalkyl, aryl, andany combination thereof, and the sulfite is a (C₁-C₆-alkyl oralkylene)-SO₃ ⁻ group; and n is 6, 7, or 8.

In some embodiments, the emulsion comprises a mixture of at least twostructurally different cyclodextrin molecules. In some embodiments, theat least two structurally different cyclodextrin molecules areindependently a compound of Formula I, wherein n is 6. In someembodiments, the at least two structurally different cyclodextrinmolecules are independently a compound of Formula I, wherein n is 7. Insome embodiments, the at least two structurally different cyclodextrinmolecules are independently a compound of Formula I, wherein n is 8.

In some embodiments, each R1 is independently H or an hydroxyalkyl,wherein at least one R1 is hydroxyalkyl. In some embodiments, each R1 isindependently H or a C₂-C₆ hydroxyalkyl, wherein at least one R1 isC₂-C₆ hydroxyalkyl. In some embodiments, each R1 is independently H or aC₂-C₆ hydroxyalkyl, wherein at least one R1 is 2-hydroxypropyl. In someembodiments, R1 is 2-hydroxypropyl. In some embodiments, thecyclodextrin molecules are 2-hydroxypropyl α-cyclodextrin.

In some embodiments, the amphiphilic polymer molecules comprise blockco-polymers, wherein the block co-polymers comprise a hydrophilic blockand a hydrophobic block. In some embodiments, the block co-polymers aredi-block co-polymers or tri-block co-polymers, or a combination thereof.In some embodiments, the block co-polymers are di-block co-polymerscomprising a polyethylene glycol (PEG) hydrophilic block and ahydrophobic block.

In some embodiments, the block co-polymers are di-block co-polymerscomprising a hydrophilic block and a hydrophobic block comprisingpolypropylene glycol. In some embodiments, the block co-polymers aredi-block co-polymers comprising a hydrophilic block and a hydrophobicblock comprising a cholesterol or a derivative thereof. In someembodiments, the block co-polymers are tri-block co-polymers. In someembodiments the tri-block co-polymers are poloxamers. In someembodiments, the poloxamers are poloxamer 188.

In some embodiments, the amphiphilic polymer molecules are non-ionicamphiphilic polymer molecules. In some embodiments, the amphiphilicpolymer molecules are linear amphiphilic polymer molecules. In someembodiments, the amphiphilic polymer molecules have a weight averagemolecular weight of from about 2,000 g/mol to about 70,000 g/mol.

In some embodiments, the droplet comprises a mass ratio of cyclodextrinmolecules to amphiphilic polymer molecules of from about 1:4 to about4:1. In some embodiments, the droplet comprises a mass ratio ofcyclodextrin molecules to amphiphilic polymer molecules of about 1:1. Insome embodiments, the droplet comprises a concentration of cyclodextrinmolecules of from about 0.5% to about 4% mass per volume (m/v). In someembodiments the concentration of cyclodextrin molecules is about 2% m/v.In some embodiments, the droplet comprises a concentration ofamphiphilic polymer molecules of from about 0.5% to about 4% mass pervolume (m/v). In some embodiments, the concentration of amphiphilicpolymer molecules is about 2% m/v. In some embodiments the cyclodextrinmolecules are at a concentration of about 2% and the amphiphilic polymermolecules are at a concentration of about 2%.

In some embodiments, the droplet further comprises a thermostableDNA-dependent DNA polymerase or an RNA-dependent DNA polymerase. In someembodiments, the droplet further comprises nucleic acid. In someembodiments, the droplet further comprises a thermostable DNA-dependentDNA polymerase or an RNA-dependent DNA polymerase. In some embodiments,the droplet further comprises: a nucleic acid; and a thermostableDNA-dependent DNA polymerase or an RNA-dependent DNA polymerase, whereinthe DNA polymerase is heterologous to the nucleic acid.

In some embodiments, the droplet comprises nucleotide triphosphates(NTPs), deoxyribonucleotide triphosphates (dNTPs), or the combinationthereof. In some embodiments, the droplet comprises a thermallyreversible hydrogel. In some embodiments, the thermally reversiblehydrogel is in a gel form at a temperature of between about 70° C. andabout 98° C. and a sol form at temperature of less than about 50° C. andgreater than about 0° C. In some embodiments, the thermally reversiblehydrogel is in a gel form at a temperature of between about 60° C. andabout 98° C. and a sol form at temperature of less than about 30° C. andgreater than about 0° C.

In some embodiments, the droplet is present in a continuous fluorocarbonoil phase. In some embodiments, the continuous fluorocarbon oil phasecomprises a fluorous oil and a surfactant. In some embodiments, thesurfactant is a fluorosurfactant. In some embodiments, thefluorosurfactant is a block co-polymer comprising a perfluorinatedpolyether block and a polyethylene glycol block. In some embodiments,the fluorosurfactant comprises a poly(perfluoro propyleneoxide)jeffamine tri-block copolymer, a poly(perfluoro propyleneoxide) amidetri-block copolymer, a poly(perfluoro propyleneoxide) PEG di-blockcopolymer, or a poly(perfluoro propyleneoxide) PEG tri-block copolymer.

In some embodiments, the fluorous oil comprises methyl nonafluorobutylether (HFE-7100), ethyl nonafluorobutyl ether (HFE-7200),2-trifluoromethyl-3-ethoxydodeca-fluorohexane (HFE-7500),1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-trifluoromethyl-pentane(HFE-7300), Perfluorooctylbromide, Perfluorodecalin, FC-40, FC-43,FC-70, FC-84, FC-72, RM-82, FC-75, RM-101, or a combination thereof. Insome embodiments, the fluorous oil is HFE-7500. In some embodiments, thedroplet is present in a continuous silicone oil phase. In someembodiments, the continuous silicone oil phase comprises a silicone oiland a surfactant.

In some embodiments, the droplet forms a contact angle of at least about90° with a cyclic olefin polymer surface after incubating the dropletfor at least about 1 hr at a temperature of about 95° C. In someembodiments, the droplet forms a contact angle of about 115° with acyclic olefin polymer surface after incubating the droplet for at leastabout 1 hr at a temperature of about 95° C. In some embodiments, theemulsion comprises at least 100, or at least 1,000 water-in-oil emulsiondroplets.

In another aspect, the present invention provides a method of forming anemulsion according to any one of the foregoing aspects or embodiments,the method comprising: combining a continuous oil phase, an aqueoussolution, a plurality of cyclodextrin molecules, and a plurality ofamphiphilic polymer molecules, under conditions wherein at least aportion of the cyclodextrin molecules and the amphiphilic polymermolecules form a plurality of polypseudorotaxane complexes, therebyforming an emulsion comprising a water-in-oil emulsion droplet. In someembodiments, the aqueous solution is a buffered aqueous solution.

In another aspect, the present invention provides a method of using anemulsion comprising a water-in-oil emulsion droplet, the methodcomprising: providing the emulsion according to any one of the foregoingemulsion aspects or embodiments; heating the droplet to a temperature ofbetween about 50° C. to about 100° C.; cooling the droplet to atemperature below about 50° C.; and optionally repeating the heating andcooling between 1-50 times. In some embodiments, after the cooling orthe optionally repeated heating and cooling, the method furthercomprises injecting at least a portion of the droplet interior into asecond droplet. In some embodiments, the droplet comprises a targetnucleic acid or an amplicon or reverse transcription product thereof. Insome embodiments, the second droplet comprises a detection reagent fordetecting the presence or absence of the target nucleic acid or theamplicon or reverse transcription product thereof.

In some embodiments, after the cooling or the optionally repeatedheating and cooling, the method further comprises injecting at least aportion of the droplet interior into a plurality of second droplets. Insome embodiments, the droplet comprises a target nucleic acid or anamplicon or reverse transcription product thereof. In some embodiments,the plurality of second droplets comprise detection reagents fordetecting the presence or absence of the target nucleic acid or theamplicon or reverse transcription product thereof. In some embodiments,the detection reagents are different in different second dropletsinjected with a portion of the first droplet, wherein the differentdetection reagents detect a presence or absence of a different targetnucleic acid or of a different amplicon or reverse transcription productthereof.

In some embodiments, the emulsion comprises at least 100 or at least1,000 water-in-oil emulsion droplets, and the method comprises:providing the emulsion comprising the at least 100 or at least 1,000water-in-oil emulsion droplets; heating the droplets to a temperature ofbetween about 50° C. to about 100° C.; cooling the droplets to atemperature below about 50° C.; and optionally repeating the heating andcooling between 1-50 times. In some embodiments, after the cooling orthe optionally repeated heating and cooling, the method furthercomprises injecting at least a portion of the droplet interiors into aplurality of second droplets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary host-guest complex between acyclodextrin and a polymer that can be used to stabilize a water-in-oilemulsion droplet described herein.

FIG. 2 shows the droplets stabilization capability and PCR compatibilityof supramolecular hydrogel formed with Pluronic F-68 and α-cyclodextrinat different mixing ratios.

FIG. 3 demonstrates the droplets stabilization capability ofsupramolecular hydrogel on Bio-Rad MonoRAyL hot-spot sequencing system.

FIG. 4 demonstrates the impact of cyclodextrin structure onsupramolecular hydrogel sample turbidity.

FIG. 5A-C illustrates results of testing water-in-oil emulsion dropletchemistries described herein having various host (cyclodextrin) andguest (polymer) concentrations and different oil compositions asindicated.

FIG. 6 demonstrates low adhesion of PCR master mix prepared fromsupramolecular hydrogel to the COP surface.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The inventors have discovered compositions that provide stablewater-in-oil emulsion droplets. These stable water-in-oil emulsionscomprise droplets in which the aqueous phase contains a cyclodextrin anda polymer capable of forming a host-guest complex with the cyclodextrin.The cyclodextrin and polymer in the droplets can form a thermallyreversible hydrogel in the aqueous phase, at the water-oil interface, orboth. The sol-gel transition temperature of the thermally reversiblehydrogel can be adjusted by altering the cyclodextrin, the cyclodextrinconcentration, the polymer, the polymer concentration, or a combinationthereof. In some cases, the stability provided by the cyclodextrin andpolymer in the aqueous phase is further enhanced with a compatible oilphase composition.

II. Compositions

Compositions provided herein comprise aqueous mixtures of cyclodextrinand polymer. In some embodiments, the aqueous mixtures provide athermally-reversible hydrogel. In some embodiments, the compositionscomprise water-in-oil (W/O) emulsion droplets that include the aqueousmixture (e.g., include an aqueous mixture that forms athermally-reversible hydrogel). In some cases, the thermally reversiblehydrogel is in a gel form at a temperature of between about 30° C. andabout 99° C., or more. In some cases, the thermally reversible hydrogelis in a gel form at a temperature of between about 40° C. and about 99°C., about 40° C. and about 95° C. or 98° C., about 45° C. and about 99°C., about 45° C. and about 95° C. or 98° C., about 50° C. and about 99°C., about 50° C. and about 95° C. or 98° C., about 60° C. and about 99°C., about 60° C. and about 95° C. or 98° C., about 70° C. and about 99°C., about 70° C. and about 98° C., or about 70° C. and about 95° C.

In some cases, the thermally reversible hydrogel is in a sol form attemperature of less than about 70° C. and greater than about 0° C., orless. In some cases, the thermally reversible hydrogel is in a sol format a temperature of between about 60° C. and about 4° C., about 50° C.and about 4° C., about 45° C. and about 4° C., about 40° C. and about 4°C., about 35° C. and about 4° C., about 30° C. and about 4° C., about25° C. and about 4° C., about 25° C. and about 10° C., about 25° C. andabout 15° C., or about 35° C., about 30° C., about 25° C., or about 20°C.

As noted above, the aqueous mixture (e.g., thermally reversible hydrogelforming aqueous mixture) can comprise a cyclodextrin and a polymercapable of forming a host-guest complex with the cyclodextrin. In someembodiments, the cyclodextrin and polymer are at a cyclodextrin:polymermass ratio of from about 0.1:1 to about 10:1, from about 0.2:1 to about5:1, from about 1:4 to about 4:1, from about 0.3:1 to about 3:1, fromabout 0.5:1 to about 2:1, or about 1:1. In some embodiments, thecyclodextrin and polymer are at a cyclodextrin:polymer molar ratio offrom about 100:1 to about 1:1, from about 50:1 to about 2:1, from about25:1 to about 3:1, from about 15:1 to about 4:1, from about 10:1 toabout 5:1, or about 7:1. In some embodiments, the cyclodextrin andpolymer are at a cyclodextrin:polymer molar ratio of from about 50:1 toabout 1:1, from about 40:1 to about 2:1, from about 30:1 to about 3:1,or from about 20:1 to about 4:1. In some embodiments, the cyclodextrinand polymer are at a cyclodextrin:polymer molar ratio of about 5:1, 6:1,7:1, 8:1, 9:1 or 10:1.

In some embodiments, the aqueous mixture (e.g., aqueous phase of a W/Oemulsion droplet) contains cyclodextrin at a concentration of from about0.1% to about 10% mass per volume (m/v), from about 0.2% to about 8%m/v, from about 0.3% to about 5% m/v, from about 0.5% to about 4% m/v,from about 1% to about 3% m/v, or about 2% m/v. In some cases, theaqueous mixture (e.g., aqueous phase of a W/O emulsion droplet) containscyclodextrin at a concentration of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, or 10% m/v. In some cases, the aqueous mixture (e.g.,aqueous phase of a W/O emulsion droplet) contains cyclodextrin at aconcentration of 2% m/v.

In some embodiments, the aqueous mixture (e.g., aqueous phase of a W/Oemulsion droplet) contains polymer (e.g., amphiphilic polymer,co-polymer, amphiphilic co-polymer, or homopolymer, as described furtherbelow) at a concentration of from about 0.1% to about 10% mass pervolume (m/v), from about 0.2% to about 8% m/v, from about 0.3% to about5% m/v, from about 0.5% to about 4% m/v, from about 1% to about 3% m/v,or about 2% m/v. In some cases, the aqueous mixture (e.g., aqueous phaseof a W/O emulsion droplet) contains polymer at a concentration of about0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% m/v. In some cases, theaqueous mixture contains polymer at a concentration of 2% m/v.

Droplets as described herein can be formed in a water-in-oil emulsion(W/O). The W/O emulsion droplets, or aqueous phase of the W/O emulsiondroplets, can be optimized to reduce or eliminate microfluidic surfacefouling by selection of appropriate parameters for the cyclodextrin andpolymer components. In some embodiments, the aqueous phase of the W/Oemulsion droplets is selected to reduce or eliminate surface fouling byminimizing or minimizing a heat-induced change in water droplet contactangle of a microfluidic surface.

In an exemplary embodiment a hydrophobic cyclic olefin polymer (COP)surface is provided, in which a pure water droplet having a volume ofapproximately 1 μL is placed on the surface. A clean and un-fouledhydrophobic COP surface can be expected to exhibit a contact angle of,of about, of at least, or of at least about 90°, 95°, 100°, 110°, or115° as measured by a goniometer. The contact angle is generallydetermined at or near room temperature. Having established that the COPsurface is hydrophobic and un-fouled, the water can be removed from theCOP surface and then contacted with a test composition under variousconditions to determine whether the composition is resistant toCOP-surface fouling.

For example, the COP surface can then be contacted with one or more, orall, components of a W/O emulsion droplet composition. In some cases,e.g., an aqueous phase of a W/O emulsion droplet as described herein iscontacted with the COP surface for a suitable period of time (e.g., 10min, 30 min, 1 hr, 3 hr, or longer, or from 10-30 min, or 10-45 min),and at a suitable temperature (e.g., at least 37° C., 40° C., 50° C.,65° C., 75° C., and/or no more than 100° C.). Typically, the temperatureis selected to approximate polymerase chain reaction (PCR) denaturationtemperature (e.g., 90° C., 95° C., or 98° C.). After testing against theemulsion droplet composition component(s), the COP surface can becleaned and dried and tested against a 1 μL droplet of pure water asdescribed above to determine whether surface fouling has occurred. Asabove, the contact angle is generally determined at or near roomtemperature. Moreover, the conditions (e.g., temperature, humidity,pressure, etc.) for the initial water droplet and the final waterdroplet are typically chosen to be the same or substantially identical.A change (e.g., reduction) in the contact angle of, of about, of lessthan, or of less than about 15%, 10%, 5%, or 1%, as compared to theinitial pure water droplet, indicates a lack of surface fouling.Generally, the smaller the change, the lower the degree of surfacefouling. Conversely a change of greater than 15%, 20%, 25%, 30%, 35%,40%, or 45% can indicate surface fouling. Generally, the greater thechange, the higher the degree of surface fouling.

Additionally or alternatively, an initial control test with a pure waterdroplet before testing the emulsion composition/components can beunnecessary when the properties of the clean COP surface arewell-characterized. Thus, a water droplet contact angle of, of about, ofat least, or of at least about 90°, 95°, 100°, 110°, or 115° aftercontacting with the aqueous phase as described above can indicate a lackof surface fouling. Conversely, a water droplet contact angle of lessthan about 90°, 80°, 70°, 60°, or 50° can indicate surface fouling.Generally the lower the contact angle, the greater the surface fouling.

In some embodiments, the W/O emulsion droplets are substantially uniformin shape and/or size. For example, in some embodiments, the droplets aresubstantially uniform in average diameter. For example, in someembodiments, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of thedroplets in the population are within 5% of the average droplet size(diameter) of the population. In some embodiments, the droplets have anaverage diameter of about 0.001 microns, about 0.005 microns, about 0.01microns, about 0.05 microns, about 0.1 microns, about 0.5 microns, about1 microns, about 5 microns, about 10 microns, about 20 microns, about 30microns, about 40 microns, about 50 microns, about 60 microns, about 70microns, about 80 microns, about 90 microns, about 100 microns, about150 microns, about 200 microns, about 300 microns, about 400 microns,about 500 microns, about 600 microns, about 700 microns, about 800microns, about 900 microns, or about 1000 microns. In some embodiments,the droplets have an average diameter of less than about 1000 microns,less than about 900 microns, less than about 800 microns, less thanabout 700 microns, less than about 600 microns, less than about 500microns, less than about 400 microns, less than about 300 microns, lessthan about 200 microns, less than about 100 microns, less than about 50microns, or less than about 25 microns. In some embodiments, thedroplets are non-uniform in shape and/or size.

In some embodiments, the droplets that are generated are substantiallyuniform in volume. For example, the standard deviation of droplet volumecan be less than about 1 picoliter, 5 picoliters, 10 picoliters, 100picoliters, 1 nL, or less than about 10 nL. In some cases, the standarddeviation of droplet volume can be less than about 10-25% of the averagedroplet volume. In some embodiments, the droplets have an average volumeof about 0.001 nL, about 0.005 nL, about 0.01 nL, about 0.02 nL, about0.03 nL, about 0.04 nL, about 0.05 nL, about 0.06 nL, about 0.07 nL,about 0.08 nL, about 0.09 nL, about 0.1 nL, about 0.2 nL, about 0.3 nL,about 0.4 nL, about 0.5 nL, about 0.6 nL, about 0.7 nL, about 0.8 nL,about 0.9 nL, about 1 nL, about 1.5 nL, about 2 nL, about 2.5 nL, about3 nL, about 3.5 nL, about 4 nL, about 4.5 nL, about 5 nL, about 5.5 nL,about 6 nL, about 6.5 nL, about 7 nL, about 7.5 nL, about 8 nL, about8.5 nL, about 9 nL, about 9.5 nL, about 10 nL, about 11 nL, about 12 nL,about 13 nL, about 14 nL, about 15 nL, about 16 nL, about 17 nL, about18 nL, about 19 nL, about 20 nL, about 25 nL, about 30 nL, about 35 nL,about 40 nL, about 45 nL, about 50 nL, or about 100 nL.

In some embodiments, the number of droplets in the W/O emulsion is, oris at least about 100; 1,000; 5,000; 10,000; 25,000; 50,000; 100,000;1×10⁶; or 1×10⁷. In some embodiments, the number of droplets is fromabout 100 to about 1×10⁷, from about 1,000 to about 1×10⁷, from about1,000 to about 1×10⁶, from about 10,000 to about 1×10⁷, from about10,000 to about 1×10⁶, from about 10,000 to about 1×10⁵, from about20,000 to about 1×10⁶, or from about 20,000 to about 1×10⁵.

The droplets can contain one or more biological analytes. Biologicalanalytes can include without limitation, cells (e.g., single cellorganisms or cells from a multicellular organism), viruses, proteins,nucleic acids (e.g., RNA or DNA, or the combination thereof), and thelike.

The droplets can contain reagents for detection of biological analytes,including but not limited to a protein such as a fluorescent proteinand/or an antibody or an enzyme such as a polymerase, peroxidase,luciferase, and/or phosphatase. In some cases, the droplets contain oneor more reagents for nucleic acid detection and/or amplification (e.g.,thermostable DNA or RNA dependent DNA polymerase, or the combinationthereof, dNTPs, buffer, salt, divalent cation, fluorescent dye,fluorescent dye-labeled nucleic acid, etc.). In some cases, a pluralityof droplets described herein contain a plurality of differentfluorescent dyes, or fluorescent dye concentrations, or a combinationthereof. The use of different fluorescent dyes and/or concentrations offluorescent dyes can provide for highly multiplex analysis.

A. Cyclodextrins

Water-in-oil (W/O) emulsion droplets described herein can contain acyclodextrin (e.g., a single chemical species of cyclodextrin molecules,albeit in numerous copies) or a mixture of different cyclodextrins.Cyclodextrins are a class of naturally occurring cyclic oligosaccharidescontaining six (α-cyclodextrin), seven (β-cyclodextrin), or eight(γ-cyclodextrin) D-glucopyranose units. Described herein are varioussuitable cyclodextrin compositions for providing a stable W/O emulsiondroplet. Moreover, it is understood that, in some cases, differentcyclodextrin compositions or mixtures thereof can be substituted tofurther optimize W/O emulsion droplet stability.

As described above, W/O emulsion droplets described herein can contain amixture of different cyclodextrins. The mixture of differentcyclodextrins in an aqueous phase of a W/O emulsion droplet can be inthe form of a randomly modified cyclodextrin (e.g., randomly methylated,randomly methacrylated, or randomly hydroxypropylated), or a mixture ofrandomly modified cyclodextrins. Alternatively, the mixture of differentcyclodextrins can be in the form of a mixture of chemically definedmodified (i.e., non-randomly modified) or unmodified cyclodextrins, or amixture of chemically defined modified and unmodified cyclodextrins. Asyet another alternative, the mixture of different cyclodextrins can bein the form of a mixture of one or more randomly modified cyclodextrinsin combination with one or more chemically defined modified orunmodified cyclodextrins. As used herein, unless otherwise indicated,references to a modified cyclodextrin or a specific species of modifiedcyclodextrin (e.g., hydroxypropyl (3-cyclodextrin) refers to a fullymodified cyclodextrin.

In some embodiments, the W/O droplets contain two or more differentcyclodextrins. In some cases, the W/O droplets contain, or contain atleast, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23 ,24, 25, 26, 27, 28, 29, or 30 different cyclodextrins. Insome cases, the W/O droplets contain from 1 to 2, from 1 to 5, from 1 to10, from 1 to 20, from 1 to 30, from 2 to 5, from 2 to 10, from 2 to 20,from 2 to 30, from 5 to 10, from 5 to 20, from 5 to 30, from 10 to 20,or from 10 to 30 different cyclodextrins.

The cyclodextrin or mixture thereof, can be or contain a modified (e.g.,randomly or non-randomly modified) or unmodified α-cyclodextrin,β-cyclodextrin, or γ-cyclodextrin, or a mixture thereof. Thecyclodextrin or mixture thereof, can be or contain a modified (e.g.,randomly or non-randomly modified) or unmodified α-cyclodextrin,β-cyclodextrin, or a mixture of modified (e.g., randomly or non-randomlymodified) or unmodified α-cyclodextrin and β-cyclodextrin. Thecyclodextrin or mixture thereof, can be or contain a modified (e.g.,randomly or non-randomly modified) or unmodified α-cyclodextrin,γ-cyclodextrin, or a mixture of modified (e.g., randomly or non-randomlymodified) or unmodified α-cyclodextrin and γ-cyclodextrin. Thecyclodextrin or mixture thereof, can be or contain a modified (e.g.,randomly or non-randomly modified) or unmodified β-cyclodextrin,γ-cyclodextrin, or a mixture of modified (e.g., randomly or non-randomlymodified) or unmodified β-cyclodextrin and γ-cyclodextrin.

Modified cyclodextrins include cyclodextrins containing one or morehydrophobic modifications. Such hydrophobic modifications include, butare not limited to methyl, ethyl, propyl, isopropyl, hydroxypropyl,butyl, C₁-C₆ or C₂-C₆ alkyl or alkylene, C₁-C₆ or C₂-C₆ hydroxyalkyl,C₁-C₆ or C₂-C₆ hydroxyalkyl acrylate, or C₁-C₆ or C₂-C₆ hydroxyalkylmethacrylate. Additionally or alternatively, the modified cyclodextrinscan contain one or more hydrophilic modifications such as C₁-C₆-alkyl oralkylene or C₂-C₆ alkyl or alkylene)-SO₃ ⁻.

In some embodiments, the cyclodextrin or mixture of cyclodextrins is acompound of Formula I or a mixture of compounds of Formula I:

wherein: each R1 is independently selected from the group consisting ofa hydrophobic group, a sulfite, and H; and n is 6, 7, or 8. In somecases, the hydrophobic group is selected from alkyl (e.g., C₁-C₆ orC₂-C₆ alkyl), alkenyl (e.g., C₁-C₆ or C₂-C₆ alkenyl), alkynyl (e.g.,C₁-C₆ or C₂-C₆ alkynyl), haloalkyl (e.g., C₁-C₆ or C₂-C₆haloalkyl),cycloalkyl (e.g., C₁-C₆ or C₂-C₆cycloalkyl), aryl (e.g., C₆ aryl), andany combination thereof. In some cases, the hydrophobic group isselected from hydroxyalkyl (e.g., C₁-C₆ or C₂-C₆ alkyl), alkyl (e.g.,C₁-C₆ or C₂-C₆ alkyl), alkenyl (e.g., C₁-C₆ or C₂-C₆ alkenyl), alkynyl(e.g., C₁-C₆ or C₂-C₆ alkynyl), haloalkyl (e.g., C₁-C₆ or C₂-C₆haloalkyl), cycloalkyl (e.g., C₁-C₆ or C₂-C₆ cycloalkyl), aryl (e.g., C₆aryl), and any combination thereof. In some cases, the sulfite is a(C₁-C₆-alkyl or alkylene)-SO₃ ⁻ group or a (C₂-C₆-alkyl or alkylene)-SO₃⁻ group.

In some cases, the emulsion (e.g., the aqueous phase of the emulsion)contains a cyclodextrin that is a compound of Formula I or contains amixture of cyclodextrins compounds of Formula I, wherein n is 6. In somecases, the emulsion (e.g., the aqueous phase of the emulsion) contains acyclodextrin that is a compound of Formula I or contains a mixture ofcyclodextrins compounds of Formula I, wherein n is 7. In some cases, theemulsion (e.g., the aqueous phase of the emulsion) contains acyclodextrin that is a compound of Formula I or contains a mixture ofcyclodextrins compounds of Formula I, wherein n is 8.

In some cases, the emulsion comprises a mixture of at least twostructurally different cyclodextrin molecules (e.g., 2 to 5, 2 to 20, or2 to 30) of Formula I, wherein n is 6. In some cases, the emulsioncomprises a mixture of at least two structurally different cyclodextrinmolecules (e.g., 2 to 5, 2 to 20, or 2 to 30) of Formula I, wherein n is7. In some cases, the emulsion comprises a mixture of at least twostructurally different cyclodextrin molecules (e.g., 2 to 5, 2 to 20, or2 to 30) of Formula I, wherein n is 8.

In some cases, each R1 is independently H or an hydroxyalkyl, wherein atleast one R1 is hydroxyalkyl. In some cases, each R1 is independently Hor a C₂-C₆ hydroxyalkyl, wherein at least one R1 is C₂-C₆ hydroxyalkyl.In some cases, each R1 is independently H or a C₂-C₆ hydroxyalkyl,wherein at least one R1 is 2-hydroxypropyl. In some cases, at least twoR1 are 2-hydroxypropyl. In some cases, at least three, at least four, orat least five R1 are 2-hydroxypropyl. In some cases, n is 7 or 8 and atleast 6 R1 are 2-hydroxypropyl. In some cases, n is 8 and at least 7 R1are 2-hydroxypropyl. In some cases, each R1 is 2-hydroxypropyl.

In some cases, the cyclodextrin component in the W/O emulsion dropletsis or comprises 2-hydroxypropyl α-cyclodextrin. In some cases, thecyclodextrin component in the W/O emulsion droplets is or comprises2-hydroxypropyl β-cyclodextrin. In some cases, the cyclodextrincomponent in the W/O emulsion droplets is or comprises 2-hydroxypropylγ-cyclodextrin.

Additional cyclodextrins include but are not limited to those describedin U.S. 2015/0025023; U.S. 2009/0012042; WO 2015/042759; U.S. Pat. Nos.5,241,059; 5,173,481, and mixtures thereof.

B. Polymers

Water-in-oil (W/O) emulsion droplets described herein also contain alinear or branched, substituted or unsubstituted, polymer or a mixtureof different polymers capable forming a host-guest complex with thecyclodextrin in the droplets. In some embodiments, the polymer is alinear polymer. In some cases, the polymer is a branched polymer, suchas a dendrimer, or a star shaped polymer. In some cases, the polymer hasa number average molecular weight or weight average molecular weight ofabout 2,000, from about 2,000 to about 70,000, or from about 7,000 toabout 10,000. In some cases, the polymer has a number average molecularweight or weight average molecular weight of from about 10 kDa to about40 kDa, from about 15 kDa to about 40 kDa, from about 10 kDa to about 70kDa, or from about 150 kDa to about 260 kDa.

In some cases, the polymer is a hydrophobic polymer (e.g., hydrophobicas compared to a linear poly(ethylene glycol) (CAS 25322-68-3) having anaverage molecular weight of 8000 (e.g., Sigma-Aldrich Product No.P5413)). In some cases, the polymer is a hydrophilic polymer, such as apoly(ethylene glycol) (PEG) polymer. In some cases, the polymer is alinear or branched PEG polymer. In some cases, the polymer is a PEGpolymer, such as a PEG described in Macromolecules 1990, 23, 2821;Macromolecules 1993, 26, 5698; Carbohydr. Res. 1998, 305, 127; orAdvanced Drug Delivery Reviews 60 (2008) 1000-1017.

In an exemplary embodiment, the PEG polymer is a star-poly(ethyleneglycol) (sPEG). For example, the PEG polymer can be an sPEG as describedin Langmuir 2003, 19, 9680-9683. As another example, the PEG polymer canbe an sPEG having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, or 24, or more arms. As another example, the PEGpolymer can be an sPEG having from 4 to 24, from 10 to 24, from 4 to 20,from 10 to 20, from 4 to 15, or from 10 to 15 arms. In some cases, thesPEG has a number average molecular weight or weight average molecularweight of about 2,000, from about 2,000 to about 70,000, or from about7,000 to about 10,000. In some cases, the sPEG has a number average orweight average molecular weight of from about 10 kDa to about 40 kDa,from about 15 kDa to about 40 kDa, from about 10 kDa to about 70 kDa, orfrom about 150 kDa to about 260 kDa. Exemplary sPEG polymers useful inthe present invention include, but are not limited to those availablefrom NEKTAR (see, 3-arm, 4-arm, and 8-arm sPEGs available fromwww.sejinbio.co.kr/Catalogue/Nektar), and/or those described inMacromolecules, 2002, 35 (5), pp 1980-1983; or Macromolecules, 2008, 41,1766-1773.

In some cases, the polymer is a polycaprolactone polymer, such as apolycaprolactone described in Macromolecules, 2000, 33, 4472-4477; orMacromolecules, 2003, 36, 2742-2747. In some cases, the polymer is apoly(propylene glycol) polymer, such as a poly(propylene glycol) polymerdescribed in J. Chem. Soc., Chem. Commun., 1990, 1322; or M.Macromolecules, 1995, 28, 8406. In some cases, the polymer is apoly(methyl vinyl ether) polymer, such as a poly(methyl vinyl ether)polymer described in Chem. Lett. 1993, 237; or Bull. Chem. Soc. Jpn.,1998, 71, 535. In some cases, the polymer is an oligoethylene polymer,such as an oligoethylene polymer described in Bull. Chem. Soc. Jpn.1994, 67, 2808.

In some cases, the polymer is a polyisobutylene polymer, such as apolyisobutylene polymer described in Macromolecules, 1993, 26, 5267; orMacromolecules, 1996, 29, 5611. In some cases, the polymer is apolylacticacid polymer, such as a polylacticacid polymer described inMacromolecules, 2003, 36, 2742-2747. In some cases, the polymer is apoly(hydroxybutyrate) polymer, such as a poly(hydroxybutyrate) polymerdescribed in Macromolecules, 2002, 35 (8), pp 3126-3132; Macromolecules,2002, 35 (9), pp 3778-3780; or Biomacromolecules, 2003November-December; 4(6):1865-7. In some cases, the polymer is apoly(tetrahydrofuran) polymer, such as a poly(tetrahydrofuran) polymerdescribed in Macromolecules, 1999, 32 (21), pp 7202-7207; J. Phys. Chem.B, 2003, 107 (1), pp 14-19; or Polymer, Volume 45, Issue 6, March 2004,Pages 1777-1785. In some cases, the polymer is a polymer of cholesterolor a derivative thereof, such as those described in Biomacromolecules,2014 Jun. 9; 15(6):2206-17; or Macromol. Chem. Phys., 2014 215: 163-170.As described herein, the foregoing polymers can be homopolymers orco-block polymers. In some cases, one or more of the monomer componentsof the foregoing polymers are suitable as a monomer or polymer block ofa block co-polymer, e.g., as further described below.

In some cases, the polymer is an amphiphilic polymer having ahydrophobic portion and a hydrophilic portion. The amphiphilic polymerscan be non-ionic amphiphilic polymers (e.g., non-ionic amphiphiliccopolymers). In some cases, the polymer is a block co-polymer (e.g., adi-block, tri-block, tetra-block, or penta-block co-polymer). In somecases, the amphiphilic polymer is a block co-polymer (e.g., a di-block,tri-block, tetra-block, or penta-block co-polymer). Amphiphilic blockco-polymers typically have at least one hydrophilic block and at leastone hydrophobic block. Exemplary hydrophilic blocks include, but are notlimited to ethylene glycol, caprolactone, lactic acid, and the like.Exemplary hydrophobic blocks include propylene glycol, hydroxybutyrate,tetrahydrofuran, isobutylene, ethylene, methyl vinyl ether, cholesterol,a cholesterol derivative, and the like.

In some cases, hydrophilic and/or hydrophobic blocks of a co-polymer aremonomer blocks. An exemplary monomer containing amphiphilic blockco-polymer is [ethyleneglycol-propyleneglycol]_(n), where n is aninteger (e.g., from 2 to 10,000). Alternatively, hydrophilic and/orhydrophobic blocks of a co-polymer are polymer blocks. Exemplary polymerblock containing amphiphilic block co-polymer include[ethyleneglycol]_(x)-[propyleneglycol]_(y), where x and y are integers,and can be the same or different (i.e.,polyethyleneglycol-polypropyleneglycol). Another exemplary polymer blockcontaining amphiphilic block co-polymer is[ethyleneglycol]_(x)-[propyleneglycol]_(y)-[ethyleneglycol]_(z), where xand y are integers, and can be the same or different (i.e.,polyethyleneglycol-polypropyleneglycol-polyethyleneglycol).

In some cases, the amphiphilic block co-polymers are tri-blockco-polymers, such as poloxamers. Poloxamers are (A-B-A) tri-blockco-polymers that contain a polyoxypropylene core block (B) flanked onboth sides by a polyoxyethylene block (A). Poloxamers are commonlyreferred to by a three digit numbering system. The first two digits x100 give the approximate molecular mass of the polyoxypropylene core,and the last digit x 10 gives the percentage polyoxyethylene content. Insome embodiments the poloxamer is a poloxamer or mixture of poloxamersdescribed by the general formula:

HO(C₂H₄O)_(a′)—[C₃H₆O]_(b)—(C₂H₄O)_(a)

wherein a′ and a can be the same or different and each is an integersuch that the hydrophilic portion represented by (C₂H₄O) constitutesapproximately 60% to 90% by weight of the copolymer, such as such as 70%to 90%, by weight, of the copolymer; and b is an integer such that thehydrophobe represented by (C₃H₆O)6 (i.e. the polyoxypropylene portion ofthe copolymer) has a molecular weight of approximately 950 to 4000daltons (Da), such as about 1,200 to 3,500 Da, for example, 1,200 to2,300 Da, 1,500 to 2,100 Da, 1,400 to 2,000 Da or 1,700 to 1,900 Da. Forexample, the molecular weight of the hydrophilic portion can be between5,000 and 15,000 Da. Exemplary poloxamers having the general formuladescribed above include poloxamers wherein a or a′ is an integer 5-150and b is an integer 15-75, such as poloxamers wherein a is an integer70-105 and b is an integer 15-75. Exemplary poloxamers include, but arenot limited to poloxamer 188, poloxamer 184, poloxamer 182, poloxamer181, poloxamer 124, poloxamer 407, poloxamer 331, and poloxamer 338.

In some embodiments, the polymer component of the cyclodextrin:polymerhost guest complex is a mixture of two or more of the foregoing polymersor mixtures thereof.

The cyclodextrin and/or polymer can be selected to form a rotaxane. Asused herein, the term “rotaxane” refers to a cyclodextrin and polymercomplex in which the polymer is threaded through the central core of thecyclodextrin and kinetically trapped from dissociation by the presenceof a bulky head group at polymer ends on each side of the threadedcyclodextrin molecule. The cyclodextrin and/or polymer can be selectedto form a pseudorotaxane. As used herein, the term “pseudorotaxane”refers to a cyclodextrin and polymer complex in which the polymer isthreaded through the central core of the cyclodextrin and relativelykinetically free to dissociate due to the absence of bulky head group atpolymer ends on each side of the threaded cyclodextrin molecule. Thepseudorotaxane complexes can be stabilized by hydrophobic non-covalentinteractions between the cyclodextrin and the polymer. Moreover,pseudorotaxane complexes can be more temperature sensitive than rotaxanecomplexes due to the absence of the stabilizing bulky head groups. Insome cases, the temperature dependence of the hydrophobic effect orother non-covalent interaction can be utilized to form pseudortoaxanecomplexes that form at relatively high temperatures, and dissociate atrelatively low temperatures. In some embodiments, this effect canprovide a thermally reversible hydrogel having a sol form at a lowtemperature and a gel form at a high temperature as described herein.

The cyclodextrin and/or polymer can be selected to form a polyrotaxane.As used herein, the term “polyrotaxane” refers to a cyclodextrin andpolymer complex in which a polymer molecule is threaded through thecentral core of multiple cyclodextrin molecules and kinetically hinderedfrom dissociation by the presence of the bulky head groups at polymerends. The cyclodextrin and/or polymer can be selected to form apolypseudorotaxane. As used herein, the term “pseudorotaxane” refers toa cyclodextrin and polymer complex in which the polymer is threadedthrough the central core of multiple cyclodextrin molecules andrelatively kinetically free to dissociate due to the absence of bulkyhead group at polymer ends on each side of the threaded cyclodextrinmolecules. The polypseudorotaxane complexes can be stabilized byhydrophobic non-covalent interactions between the cyclodextrin and thepolymer. Moreover, polypseudorotaxane complexes can be more temperaturesensitive than polyrotaxane complexes due to the absence of thestabilizing bulky head groups. In some cases, the temperature dependenceof the hydrophobic effect or other non-covalent interaction can beutilized to form polypseudorotaxane complexes that form at relativelyhigh temperatures, and dissociate at relatively low temperatures. Insome embodiments, this effect can provide a thermally reversiblehydrogel having a sol form at a low temperature and a gel form at a hightemperature as described herein.

C. Oil Phase

The W/O emulsion droplets are present in a continuous oil phase.Typically, the continuous oil phase contains a fluorous oil, a siliconoil, or a combination thereof. Non-limiting examples of fluorous oilssuitable for use as a continuous oil phase or in a continuous oil phaseinclude methyl nonafluorobutyl ether (HFE-7100), ethyl nonafluorobutylether (HFE-7200), 2-trifluoromethyl-3-ethoxydodeca-fluorohexane(HFE-7500),1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-trifluoromethyl-pentane(HFE-7300), perfluorooctylbromide, perfluorodecalin, FC-40, FC-43,FC-70, FC-770, FC-84, FC-72, RM-82, FC-75, RM-101, or a combinationthereof.

The continuous oil phase can also contain a surfactant, such as afluorosurfactant. In some cases, the fluorosurfactant is perfluorinatedor is a block co-polymer that contains a perfluorinated block. Exemplaryfluorosurfactants suitable for use in a continuous oil phase include,but are not limited to, Krytox fluorosurfactants such as Krytox FSH(Krytox FSH 157), fluorosurfactants described in U.S. Pat. No.9,012,390, and mixtures thereof. The surfactant in the continuous oilphase can be present at a concentration of from about 0.01% to about 5%,from about 0.1% to about 4%, from about 0.5% to about 3%, from about 1%to about 3%, or about 2% (w/w). In some embodiments, the oil phasecomprises an ionic fluorosurfactant. In some embodiments, the ionicfluorosurfactant is Ammonium Krytox (Krytox-AS), the ammonium salt ofKrytox FSH, or a morpholino derivative of Krytox FSH. Krytox-AS can bepresent at a concentration of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). In some embodiments,the concentration of Krytox-AS is about 1.8%. In some embodiments, theconcentration of Krytox-AS is about 1.62%. Morpholino derivative ofKrytox FSH can be present at a concentration of about 0.1%, 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, or 4.0% (w/w). Insome embodiments, the concentration of morpholino derivative of KrytoxFSH is about 1.8%. In some embodiments, the concentration of morpholinoderivative of Krytox FSH is about 1.62%.

In some embodiments, the oil phase further comprises an additive fortuning the oil properties, such as vapor pressure, viscosity, or surfacetension. Non-limiting examples include perfluorooctanol and1H,1H,2H,2H-Perfluorodecanol. In some embodiments,1H,1H,2H,2H-Perfluorodecanol is added to a concentration of about 0.05%,0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9%, 1.0%, 1.25%, 1.50%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, or 3.0%(w/w). In some embodiments, 1H,1H,2H,2H-Perfluorodecanol is added to aconcentration of about 0.18% (w/w).

III. Methods

Described herein are methods of making one or more of the foregoing W/Oemulsion droplet compositions. Typically, the method includes combiningthree or more aqueous phase components (e.g., water, cyclodextrin, andpolymer) to form an aqueous reaction mixture and then combining theaqueous reaction mixture with one or more oil phase components (e.g.,fluorous and/or silicone oil and optionally fluorosurfactant) to form anemulsion pre-mixture. The pre-mixture can be emulsified using methodsknown in the art to form a plurality of W/O emulsion droplets. Forexample, the emulsion pre-mixture can be sonicated, vortexed, blended,or a combination thereof to form the W/O emulsion droplets.Alternatively, the aqueous reaction mixture and oil phase components canbe combined using methods to directly generate a plurality of W/Oemulsion droplets without an intervening emulsion pre-mixture. Forexample, a microfluidic device can be used to generate emulsion dropletsin a continuous oil phase by injecting a plurality of droplet-sizevolumes of aqueous reaction mixture into the continuous oil phase.

As yet another alternative, W/O emulsion droplets can be generated bycombining a continuous oil phase (e.g., a base oil and optionally afluorosurfactant), an aqueous solution (e.g., water or buffered water),a plurality of cyclodextrin molecules, and a plurality of (e.g.,amphiphilic) polymer molecules, under conditions wherein at least aportion of the cyclodextrin molecules and the polymer molecules form aplurality of host-guest complexes (e.g., rotaxane, pseudorotaxane,polyrotaxane, or polypseudorotaxane complexes), thereby forming anemulsion comprising a water-in-oil emulsion droplet. As described above,the combining to form the emulsion can be performed in bulk (e.g., withsonicating, vortexing, blending, or a combination thereof) or byinjecting a plurality of droplet-size volumes of aqueous reactionmixture into the continuous oil phase. In some cases one or morecomponents of the aqueous phase, one or more components of thecontinuous phase, or a combination thereof, are not included in theemulsification, but added at a later time, e.g., by injection of thecomponent into the droplet, or by merging droplets. Methods,compositions, devices, and systems for injecting or merging componentsinto a droplet or a plurality of droplets include but are not limited tothose described in U.S. 2015/0209785; U.S. 2016/0131675; U.S.2015/0321163; U.S. 2013/0344485; U.S. 2015/0024945; U.S. 2015/0045258;U.S. 2015/0065396; U.S. 2016/0001289; and U.S. 2016/0045914, each ofwhich are incorporated by reference in the entirety for any and allpurposes including but not limited to droplet compositions, dropletmethods, and devices and systems for making and/or using droplets foranalysis of biological analytes such as nucleic acids.

Also described herein are methods of using a W/O emulsion containing aW/O emulsion droplet (e.g., a plurality of W/O emulsion droplets). Insome embodiments, the emulsion droplets are used in a microfluidicdevice. Additionally or alternatively, the droplets can be used in adevice configured to perform bulk analysis. For example, a plurality ofW/O emulsion droplets can be introduced into a PCR tube (e.g., a 0.2 mLPCR tube) and thermocycled in a thermocycler to, e.g., amplify and/ordetect nucleic acids. In some embodiments, the emulsion droplets canthen be analyzed in a microfluidic device. Alternatively, the emulsiondroplets can be thermocycled and analyzed, or generated, thermocycled,and analyzed in a microfluidic device.

In one embodiment, described herein is a method of using a W/O emulsiondroplet by providing the emulsion containing the W/O emulsion droplet;heating the droplet to a temperature of between about 50° C. to about100° C.; cooling the droplet to a temperature below about 50° C.; andoptionally repeating the heating and cooling between 1-500, 1-250,1-100, 1-50, 1-40, 10-500, 10-250, 10-100, 10-40, 20-500, 20-250,20-100, 20-50, or 20-40 times. In some cases, the method includesheating to a nucleic acid denaturation temperature (e.g., about 95° C.,98° C., or 99° C.), cooling to a nucleic acid primer hybridizationand/or polymerization temperature (e.g., about 50° C., 60° C., 65° C.,68° C., 70° C., or 72° C.), optionally heating to a nucleic acid primerpolymerization temperature; and optionally repeating the denaturation,hybridization/polymerization, and optional polymerization temperaturesbetween 1-500, 1-250, 1-100, 1-50, 1-40, 10-500, 10-250, 10-100, 10-40,20-500, 20-250, 20-100, 20-50, or 20-40 times.

In some embodiments at least 100 or at least 1,000, or more W/O emulsiondroplets are thermocycled, e.g., as described herein. In someembodiments, after the cooling or repeated heating and cooling of theW/O emulsion droplets, the method further includes injecting at least aportion of the droplet interiors into a plurality of second droplets(e.g., second droplets containing detection reagents).

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention. Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

EXAMPLES

Droplet Stabilization with Poloxamer:Cyclodextrin Emulsions

The poloxamer Pluronic F-68 was tested with α-cyclodextrin at variousmixing ratios to identify compositions which formed suitablethermally-reversible host-guest complexes (see, e.g., FIG. 1) and/or,e.g., thermally reversible, supramolecular hydrogels. In thisexperiment, the Pluronic F-68 and α-cyclodextrin were added to abuffered aqueous PCR master mix comprising KRAS amplicon, DNApolymerase, primers and dNTPs at (8% m/v+2% m/v) and (1% m/v+1% m/v)final concentration, respectively. The droplets containing the PCRmaster mix were generated by a microfluidic flow-focusing device with an85 μm nozzle. The oil phase comprised HFE7500 with 2% w/wKrytox-Jeffamine-Krytox based triblock copolymer as fluoro-phasesurfactant. Upon generation, droplets-oil mixtures comprising 70-90 μLdroplets and 30-10 μL oil were transferred to 200 μL PCR tubes at atotal volume of 100 μL per tube then thermal cycled on Bio-Rad MyCycler™thermal cycler.

After thermal cycling, the droplets were visually inspected in bulk(FIG. 2a, 2d ) and on Bio-Rad ZOE™ Fluorescent Cell Imager (FIG. 2b ) tocapture any coalescence instance. FIG. 2a, 2b, 2d demonstrated that thedroplets stabilized with supramolecular hydrogel were highlymonodispersed with minimum coalescence post thermal cycling.

The droplets were broken by mixing with 1H,1H,2H,2H-Perfluoro-1-octanolat 1:1 volume ratio for gel electrophoresis analysis. The gelelectrophoresis demonstrated that the supramolecular hydrogel additiveis compatible with PCR (FIG. 2c, 2e ).

Droplets Stabilization Capability of Supramolecular Hydrogel on Bio-RadMonorayl Hot-Spot Sequencing System

In this experiment, the Pluronic F-68 and 2-hydroxypropyl-α-cyclodextrinwere added to a buffered aqueous PCR master mix comprising KRASamplicon, DNA polymerase, primers and dNTPs at 2% m/v+2% m/v finalconcentration. The droplets containing the PCR master mix were generatedand thermal cycled with Bio-Rad MonoRAyL hot-spot sequencing system.FIG. 3 shows highly monodispersed droplets post on-chip thermal cycling.

Supramolecular Hydrogel Sample Turbidity Dependence on CyclodextrinMolecular Structure

In this experiment, aqueous PCR master mix were prepared with 2%Pluronic F-68+2% α-cyclodextrin and 2% Pluronic F-68+2%2-hydroxypropyl-α-cyclodextrin, respectively and the mastermix solutionswere stored at 4° C. for two weeks. The sample prepared fromα-cyclodextrin (top image) showed increased turbidity upon prolongedstorage while the sample prepared from 2-hydroxypropyl-α-cyclodextrin(bottom image) still remained clear.

Droplets Stabilization Capability and PCR Compatibility ofSupramolecular Hydrogel Formed with Pluronic F-68 and2-Hydroxypropyl-A-Cyclodextrin at Different Concentrations and withDifferent Oil-Phase Surfactants

In this experiment, the Pluronic F-68 and 2-hydroxypropyl-α-cyclodextrinwere added to a buffered aqueous PCR master mix comprising KRASamplicon, DNA polymerase, primers and dNTPs at (0.5% m/v+0.5% m/v), (1%m/v+1% m/v), (2% m/v+2% m/v) and (4% m/v+4% m/v) final concentration,respectively. The droplets containing the PCR master mix were generatedby a microfluidic flow-focusing device with 85 μm nozzle. The oil phasecomprised HFE7500 with 2% w/w Krytox-PEG based diblock copolymer asfluoro-phase surfactant. Upon generation, droplets-oil mixturescomprising 70-90 μL droplets and 30-10 μL oil were transferred to 200 μLPCR tubes at a total volume of 100 μL per tube then thermal cycled onBio-Rad MyCycler™ thermal cycler. After PCR, the emulsions were visuallyinspected in bulk. FIG. 5a shows that emulsions prepared with differentconcentration of supramolecular hydrogel retained good stability postthermal cycling. The droplets were broken by mixing with 1H,1H,2H,2H-Perfluoro-1-octanol at 1:1 volume ratio for gel electrophoresisanalysis. The gel electrophoresis demonstrated that the supramolecularhydrogel additive is compatible with PCR at the tested concentrationrange (FIG. 5b ).

Similarly, Pluronic F-68 and 2-hydroxypropyl-α-cyclodextrin were addedto a buffered aqueous PCR master mix comprising KRAS amplicon, DNApolymerase, primers and dNTPs at 2% m/v+2% m/v final concentration. Thedroplets containing the PCR master mix were generated by a microfluidicflow-focusing device with 85 μm nozzle. The oil phase comprised HFE7500with 2% w/w of different types of fluoro-phase surfactant: 1.Krytox-PEG-Krytox based triblock copolymer with amide linkage; 2.Krytox-PEG based diblock copolymer with ester linkage; 3.Krytox-PEG-Krytox based triblock copolymer with ester linkage; and 4.Krytox-Jeffamine-Krytox based triblock copolymer with amide linkage.Upon generation, droplets-oil mixtures comprising 70-90 μL droplets and30-10 μL oil were transferred to 200 μL PCR tubes at a total volume of100 μL per tube then thermal cycled on Bio-Rad MyCycler™ thermal cycler.After PCR, the emulsions were visually inspected in bulk. FIG. 5c showsthat emulsions prepared with supramolecular hydrogel exhibit goodthermal stability with different types of fluoro-phase surfactant. Thedroplets without supramolecular hydrogel were prepared as negativecontrol and they showed severe coalescence post thermal cycling.

Low Adhesion of PCR Master Mix Prepared from Supramolecular Hydrogel tothe COP Surface Demonstrates Resistance to Surface Fouling

In this experiment, the Pluronic F-68 and 2-hydroxypropyl-α-cyclodextrinwere added to a buffered aqueous PCR master mix comprising KRASamplicon, DNA polymerase, primers and dNTPs at 2% m/v+2% m/v finalconcentration. The master mix without supramolecular gel but containing0.1 mg/ml BSA was prepared as negative control. A small plaque ofhydrophobically coated COP sample was submerged into master mix and washeated at 95° C. in a sealed vial. The contact angle of the COP plaquewas measured to capture any sample adhesion to the surface. FIG. 6 showsthat the contact angle of COP maintained same over 5 hours incubation at95° C. in the supramolecular hydrogel master mix, indicating minimumsample adhesion to the surface. In contrast, the COP plaque exposed to0.1 mg/ml BSA master mix shows rapid decrease in contact angle,indicating significant sample adhesion to the surface.

What is claimed is:
 1. An emulsion comprising a water-in-oil emulsion droplets, the droplets comprising: water; a plurality of cyclodextrin molecules, wherein at least some of the cyclodextrin molecules comprise 2-hydroxypropyl α-cyclodextrin, 2-hydroxypropyl β-cyclodextrin, or 2-hydroxypropyl γ-cyclodextrin; and a plurality of amphiphilic polymer molecules, wherein individual amphiphilic polymer molecules of the plurality comprise a hydrophobic moiety and a hydrophilic moiety, wherein the amphiphilic polymer molecules are poloxamers; wherein at least a portion of the cyclodextrin molecules and the amphiphilic polymer molecules form a thermally-reversible hydrogel that is in sol form at at least one temperature between 0-70° C., wherein the thermally-reversible hydrogel comprises a plurality of polypseudorotaxane complexes, and wherein the droplets are present in a continuous oil phase.
 2. The emulsion of claim 1, wherein a mass ratio of cyclodextrin molecules to amphiphilic polymer molecules in the droplets is from about 1:4 to about 4:1.
 3. The emulsion of claim 1, wherein a mass ratio of cyclodextrin molecules to amphiphilic polymer molecules in the droplets is 1:1.
 4. The emulsion of claim 1, wherein the concentration of cyclodextrin molecules in the droplets is from 0.5% to 4% mass per volume (m/v).
 5. The emulsion of claim 4, wherein the concentration of cyclodextrin molecules is 2% mass per volume (m/v).
 6. The emulsion of claim 4, wherein the poloxamers are poloxamer
 188. 7. The emulsion of claim 1, wherein the cyclodextrin molecules comprise 2-hydroxypropyl α-cyclodextrin.
 8. The emulsion of claim 1, wherein the cyclodextrin molecules comprise 2-hydroxypropyl β-cyclodextrin.
 9. The emulsion of claim 1, wherein the cyclodextrin molecules comprise 2-hydroxypropyl γ-cyclodextrin.
 10. The emulsion of claim 1, wherein the poloxamers are poloxamer
 188. 11. The emulsion of claim 1, wherein the droplets further comprise a thermostable DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase.
 12. The emulsion of claim 1, wherein the droplets further comprise nucleic acid.
 13. The emulsion of claim 1, wherein the droplets comprise a thermally reversible hydrogel.
 14. The emulsion of claim 1, wherein the droplets are present in a continuous fluorocarbon oil phase.
 15. The emulsion of claim 14, wherein the continuous fluorocarbon oil phase comprises a fluorous oil and a surfactant.
 16. The emulsion of claim 15, wherein the surfactant is a fluorosurfactant.
 17. The emulsion of claim 16, wherein the fluorosurfactant is a block co-polymer comprising a perfluorinated polyether block and a polyethylene glycol block.
 18. A method of forming an emulsion according to claim 1, the method comprising: combining a continuous oil phase, an aqueous solution, a plurality of cyclodextrin molecules, and a plurality of amphiphilic polymer molecules, under conditions wherein at least a portion of the cyclodextrin molecules and the amphiphilic polymer molecules form a plurality of polypseudorotaxane complexes, thereby forming an emulsion comprising a water-in-oil emulsion droplets.
 19. A method of using an emulsion comprising a water-in-oil emulsion droplets, the method comprising: providing the emulsion according to claim 1; heating the droplets to a temperature of between about 50° C. to about 100° C.; cooling the droplets to a temperature below about 50° C.; and optionally repeating the heating and cooling between 1-50 times. 